Much experimental effort has been directed to the study of RBC in sickle cell disease, but as yet the precise pathogenic mechanisms that connect the single hemoglobin abnormality to the multiple membrane and cytoplasmic defects seen in the RBC remain obscure. It is clear that the pathophysiology is complex, and that complexity is compounded further by the heterogeneity of RBC in the blood of any individual patient, and also by the heterogeneity of genetic factors among patients. A central hypothesis of this project is that by studying cells which are precisely defined both by age (i.e., reticulocytes) and by genotype )beta/s haplotypes and alpha-gene status), it will be possible to eliminate much of the heterogeneity that can confound the interpretation of studies on whole blood from patients of variable genotype. In this way, we hope to identify some of the intrinsic differences in RBC that underlie the variability of clinical severity in sickle cell disease patients, and to clarify mechanisms that lead to the cellular changes in sickle cell disease. This project brings together three approaches in which this Center has expertise: 1) extensive previous research into the association of specific beta/s gene cluster haplotypes and alpha-gene deletions with the severity of clinical disease and degree of hematological abnormality; 2) the development of specialized rheological techniques and instrumentations to enable the study of very small quantities of blood and small subpopulations of cells; and 3) the ability the produce erythrocytes in vitro from early hematopoietic progenitor cells--'virgin' RBC which have not been exposed to the rigors of passage through circulation. The specific objectives of this project are: To determine whether the hematologic and rheologic changes associated with different beta/s haplotypes are due to specific differences intrinsic to the RBC; to study the consequences of sickling in cultured RBC that have never previously been deoxygenated and to compare the results across different beta/s haplotypes; and to test the hypothesis that the variable clinical severity associated with different beta/s haplotypes may be due to a subpopulation of reticulocytes with minimal resistances to deoxygenation and dehydration.